10TH INTERNATIONAL SYMPOSIUM ON PARTICLE IMAGE VELOCIMETRY – PIV13 Delft, The Netherlands, July 2-4, 2013
Measurement of Three-Dimensional Velocity upon Endothelial Cells
Utilizing Confocal Micro-PIV
Yasuhiko Sugii 1, Takuya Mukouyama 2 and Koichi Hishida 2
1
School of Engineering, the University of Tokyo, Tokyo, Japan tsugii@mail.ecc.u-tokyo.ac.jp
2
Department of System Design Engineering, Keio University, Yokohama, Japan
ABSTRACT
To investigate an influence of a glycocalyx surface layer covering over endothelial cells (ECs) on the flow field or flow through the layer, a high spatial resolution measurement technique was improved for three-dimensional velocity distributions close to a glycocalyx layer by utilizing a confocal micro-PIV (Particle Image Velocimetry) technique. The developed technique achieved to measure three-dimensional velocity distributions in a 177 × 177 × 3 m3 region with
in-plane resolution of 7 m and with depth resolution of 0.5 m. By optimizing the PIV analysis, the measurement accuracy was improved even with small fluorescent particle of the diameter of 200 nm and large shear rate. Three-dimensional velocity distribution close to the layer of living ECs cultured in microchannel was measured. The results suggested that the developed technique was useful for investigation of velocity field close to the layer.
INTRODUCTION
Blood flow plays a critical role in maintaining homeostasis of tissue and organs such as delivering oxygen and nutrients. To investigate cause of vascular diseases such as cerebral infarction and cardiac infarction, a number of studies for biophysical behavior of blood flow in the vascular system have been carried out [1-4]. Fluid shear stress was related to physiological vascular functions such as maintenance of vessel tone, prevention of thrombosis and initiation of angiogenesis [5, 6]. Secretion of nitric oxide (NO) and intercellular calcium (Ca2+), and remodeling of blood vessel
such as elongation and orientation in the flow direction were observed as response of Endothelial cells (ECs), which line inner surface of blood vessel, to fluid shear stress [7, 8]. One of the important factors in determining biophysical behavior of blood flow was considered to be glycocalyx surface layer [9, 10]. The thickness of the layer was measured using the transmission electron microscopy (TEM) and varied from 20 nm, 200 -500 nm to 2 - 4.5 μm [11-14]. Blood flow resistance and vascular permeability, such as limiting access of certain molecules to the EC membrane, were notably affected by existence or non-existence of the layer [15- 16]. Morphological change and production of NO was also affected by the layer [17]. The layer was considered to play an important role in sensing of wall shear stress and in mechanotransduction [18]. Therefore, to clarify biophysical behavior of blood flow, it is important to investigate the flow field in vicinity of cell surface especially near the layer.
In order to investigate the influence of the layer on the flow field or flow through the layer, various theoretical studies have been carried out [19-25]. Since the glycocalyx layer shows bush like structure and elasticity, the layer has been modeled as a biphasic mixture, or porous layer, with a linearly elastic solid phase and an incompressible Newtonian fluid phase. These models indicated that the glycocalyx lead to lower fluid wall shear stresses at the endothelial surface than in the absence of the layer. Even a relatively thin glycocalyx with sufficiently low permeability impeded fluid flow near vessel wall and exerted a significant influence on microvascular resistance. The glycocalyx behaves as a molecular filter of water, ions and small hydrophilic solutes between the plasma and cells. Conversely, a few experimental studies have been reported due to difficulty of measurement in its small scale. Conventional micro particle image velocimetry (micro-PIV) technique was applied to flow field near the vessel wall in post-capillary venules in vivo [26]. The authors developed a high spatial resolution measurement technique for velocity distributions close to surface of cell utilizing confocal micro-PIV technique [27]. However, these measurement techniques were insufficient in a spatial resolution and accuracy to investigate the influence of the layer.
In the study, in order to investigate an influence of a glycocalyx surface layer on a flow field, a high spatial resolution measurement technique was improved for three-dimensional velocity distributions close to a glycocalyx layer by utilizing confocal micro-PIV technique. The optics was optimized and the PIV analysis was improved to obtain three-dimensional velocity distribution with high accuracy close to the layer by scanning very thin measurement plane. Three-dimensional velocity distribution near the layer of living ECs cultured in microchannel was investigated.
EXPERIMENTAL SETUP
Figure 1 shows the developed confocal micro-PIV system, which enables to remove out-of-focus light optically and obtain higher contrast image than conventional micro-PIV system [28], in order to improve a spatial resolution and measurement accuracy. The system consisted of an inverted microscope with a dual Nipkow disk-type confocal scanner, CSU-X1 (Yokogawa Elc. Cop., Japan), a CW laser (SAPPHIRE; Coherent) with a wavelength = 488 nm and a high speed camera (SV-200i; Photron, 10bit) set 1000 fps with resolution of 512 × 512 pixels. By using a high numerical aperture lens, a 40× oil immersion objective lens (Plan Fluor; Nikon, NA = 1.3), and a piezo actuator (P-721K120; PI) with sub-nm resolution accuracy, three dimensional velocity distributions with high spatial resolution was achieved. The depth of focus of the confocal optical system was estimated to be 0.58 m. The measurement volume was corresponded to 177 × 177 × 3 m3, in which depth resolution was 0.5 m from Z = 13 to Z = 15.5 m as shown in
figure 2. Culture medium seeded with fluorescent particles with a diameter of 200 nm, which excites at 505 nm and emits at 515 nm, was used as a working fluid. The fluid was injected by using a microsyringe pump at the constant flow rate of 3.0 L/min, Reynolds number Re = 0.21, which corresponded with flow rate of in vivo condition.
Figure 1 Schematic diagram of confocal micro-PIV system.
Figure 2 Measurement volume for velocity distributions around endothelial cell scanned by piezo actuator.
Since a dual Nipkow disk-type confocal scanner provides that an exposure time of a frame was almost equal to inverse of the frame rate, it is necessary to set enough higher frame rate compared with particle displacement to avoid a long streakline, which causes low measurement accuracy. Therefore high frame rate, 1000 fps, for all of focal planes compared with particle displacement was settled. To improve measurement accuracy by optimizing particle displacement, image pair from sequential images was settled varying the time interval t with focal position shown as figure 3. Appling the cross-correlation method and a Gaussian peak fitting method for sub-pixel analysis with an interrogation window of 40 × 40 pixels with 50% overlap, which was corresponded with in-plane spatial resolution of 7 × 7 m2, 200 velocity distributions over all image pair were obtained. Temporal averaging was applied over 200
velocity distributions to eliminate the effect of Brownian motion. The time interval at each focal plane at the minimum standard deviation was used for PIV analysis.
Z
VelocityY
Objective lens Piezo actuator Endothelial cell 13 - 14 m Plane 1 (Z = 13.0 m) Plane 2 (Z = 13.5 m) Plane 6 (Z = 15.5 m) Plane 3 (Z = 14.0 m) Plane 4 (Z = 14.5 m) Plane 5 (Z = 15.0 m)Figure 3 Optimization of PIV analysis by adjusting the time interval of image pairs.
Figure 4 Fluorescent particle image around the microchannel bottom with endothelial cells.
Flow field around living ECs cultured in a microchannel as shown in figure 4 (a) and (b) was used. A polydimethylsiloxane (PDMS) microchip consists of a single straight microchannel, which has a rectangular cross sectional shape with width of 400 m, depth of 120 m and long of 2 cm fabricated using soft-lithographic techniques as follows. A negative photoresist SU-8 was poured on a glass plate using spin-coater. After soft-bake on a hot plate, UV-light was exposed through a photolithography mask using a mask aligner to impress the channel pattern onto the SU-8. The exposed substrate was hard-baked and developed with a developer solution to remove photoresist in unexposed areas. A PDMS slurry on the master was baked in the oven. The glass slide with thickness of 170 m was attached to the PDMS. Microtubes with the inner diameter of 500 m were connected to microchip for fluid inlet and outlet. Human umbilical vein endothelial cells (HUVECs), which cultured in a 35 mm2 dish with an EC culture medium
(EGM-2, Lonza), were used in the experiments. After sterilization using ultraviolet lump and 70 % ethanol, the microchannel wall was coated with matrigel, which is one of an extracellular matrix gel and often used for surface coating of a dish. Solution of matrigel (0.1 mg/mL) was introduced into the microchannel. The microchip was incubated in a 5 % CO2 incubator at 37 degree Celsius for 1 hour without flow to adsorb matrigel and then rinsed with the culture
medium. ECs suspension at a cell density of 106 cells/mL was introduced at the flow rate of 5.0 l/min by withdrawing
the micro syringe by the pump shown. The microchip was incubated in the 5 % CO2 incubator at 37 degree Celsius for
2 hours to be attached ECs on the inner wall without flow. Figure 4 (c) shows a fluorescent particles image at the bottom of the microchannel, Z = 0 m. White vertical line in the figure represents the side wall of the microchannel.
Red dashed lines represents ECs with size of 30 - 50 m, in which fluorescent particle was absent. The culture medium flowed in the microchannel from bottom to top in the figure.
RESULTS
Figure 7 shows three-dimensional temporal averaged velocity distributions at six measurement planes with depth resolution of 0.5 m in the microchannel with ECs obtained by the proposed technique after the optimization process as mention above. Height of the top of EC was roughly estimated to be 13 m around X = 120, Y = 80 m in the figure. Velocity vectors at all of the planes were almost parallel to the side wall, and velocity gradually increased from the side wall to the center of the channel. Maximum velocity was 0.76 mm/s around the center of the channel at Z = 15.5 m. Velocities were corresponded with theoretical value of laminar flow in the microchannel except them of upper parts of ECs. Velocities of upper parts of ECs were smaller than theoretical value. These velocities show the effect of the EC shape. The results indicated the developed technique was achieved high accuracy and high spatial resolution in spite of the large shear rate and close to wall.
Figure 5 Three dimensional temporal averaged velocity distributions at six planes.
CONCLUSIONS
We improved a high resolution measurement technique for three-dimensional velocity distributions close to a glycocalyx surface layer of living ECs by utilizing confocal micro-PIV to investigate the flow field in vicinity of vessel wall. The developed technique enabled to measure three-dimensional velocity distributions of micro-flows in a 177 × 177 × 3 m3 region with in-plane resolution of 7 m and with depth resolution of 0.5 m. By optimizing the PIV
analysis with adjusting the time interval, the measurement accuracy was improved even with small fluorescent particle of the diameter of 200 nm and large shear rate. Three-dimensional velocity distributions around living ECs culture in the PDMS microchannel with the rectangular cross sectional shape, width of 400 m and depth of 120 m were measured at six planes spaced 0.5 m apart. The obtained velocity distributions indicated the influence of the EC on the flow field. The results showed the developed technique was achieved high accuracy and high spatial resolution in spite of the large shear rate and close to wall.
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